1. Field of the Invention
This invention relates to a method for assaying genetic sequences to genetic sequences useful in the method, and to the relevant components of a kit for the assay of such sequences. It also embodies the selection and use of triple helix forming regions or triple strand forming regions in a number of genes of clinical importance.
The identification of genetic sequences is an important procedure in biological, medical and biotechnological research. Quantitative information about the copy number of genes in humans is particularly important in the early recognition of cancer and the prenatal identification of genetic defects. In the case of cancer cell recognition, such cells frequently have elevated copy numbers of regulatory genes and in some cases, the severity of the prognosis reflects the magnitude of the increase in copy number. Early detection while the copy number is still relatively low allows treatments that can give complete elimination of the cancer with minimum discomfort to the patient. The prenatal detection of genetic defects such as trisomy (three copies of a particular chromosome) or deletions or duplications of small portions can prepare parents and medical staff for later problems and the screening of high risk groups for such defects can help limit the number of births of children with low survival.
2. Description of the Related Art
A number of techniques exist for the detection of genes in human DNA but all of these are skilled techniques that are still principally used in the research laboratory environment. Southern blotting, Polymerase Chain Reaction and Ligase Chain Reaction have all been used to detect specific DNA sequences, but making the techniques quantitative is an extremely skilled process requiring considerable technical expertise and some statistical analysis. The technique described herein is part of a larger process that offers an easy-to-use alternative that combines sensitivity with reliability.
Triple helix formation is described in a publication of Moser, H. E. and Dervan, P. B. (1987) Science, Vol. 238, pages 645-650.
European Publication EP-A 0 330 185 of the present assignee describes a method for assaying genetic sequences in which a labelled reporter molecule is hybridized with a genetic probe and upon introduction of genetic material which more strongly hybridizes with the genetic probe, the reporter molecule disassociates from the genetic probe and is detected.
European Publication EP-A 0 304 845 of the present assignee describes and claims a method of assaying gene expressions which comprises linking MRNA molecules to a solid substrate and contacting the solid substrate with an aqueous suspension of labelled microbeads to which gene probe molecules are linked. The gene probe molecules comprise sequences which hybridize with sequences comprised by and characteristic of the unknown or target mRNA molecules to be assayed, so that the labelled microbeads are linked to the target MRNA molecules on the substrate. By separating the solid substrate from microbeads unlinked to target mRNA molecules and detecting labelled, linked microbeads, the gene expressions are assayed.
Labelled microbeads have been used in a number of analytical methods such as immunoassay methods. Extensive prior art concerning microbeads is described in U.S. Pat. Nos. 4,454,233 granted Jun. 12, 1984 and 4,436,826 granted Mar. 13, 1984, both assigned to the present assignee.
The method of the present invention for assaying genetic sequences is based on displacement of a reporter molecule hybridized to an anchored single-stranded DNA molecule. The displacement relies on the formation of a triple helical or triple strand DNA structure (triplex) between a portion of the sequence of the reporter and double-stranded DNA sequences in sample DNA. The reporter molecule in this case constitutes the "third strand" of the triple helix. The reporter/anchor hybrid is maintained in a suitable buffer in a reaction vessel. Double-stranded sample DNA is added to the buffer and the whole is incubated at a defined temperature. The free 5'-end of said reporter molecule forms a triple helical DNA segment with the sample DNA. The formation of the triple helix reduces the stability of the reporter/anchor stability and thus the reporter is displaced from the anchor DNA to the liquid phase where it can be assayed by means of the submicron particle attached to the reporter. The steps in this process are shown in diagrammatic form in FIG. 1.
According to the present invention there is provided a method for assaying genetic sequences which comprises the formation of a weak DNA-DNA duplex in aqueous solution where one of the strands (the anchor strand) is covalently bound to a solid phase and the other (the reporter strand) has an assayable molecule or particle comprising assayable groups attached to one end. A portion of the reporter molecule is not hybridized to the anchor molecule and has a DNA sequence such that it can form a triple-helical DNA structure with a double stranded duplex DNA molecule of suitable sequence. This double stranded DNA molecule would be found in the sample DNA added to the reaction vessel. When the triple helical DNA structure forms, it causes the reporter molecule to be displaced from the reporter/anchor duplex. The amount of said reporter molecule that is displaced, when compared with suitable standards, will then give a measure of the amount of the double helical sequences present in the sample. The use of specific DNA sequences allows for absolute specificity in the displacement reaction.
According to the present invention there is provided a method of assaying genetic sequences, which comprises introducing a sample containing double stranded DNA to be assayed into an aqueous medium containing at least one complex comprising an anchor DNA strand anchored to a support matrix, said anchor strand being hybridized with a reporter DNA strand having a detectable label, a portion of the anchor strand or the reporter strand consisting of a selected sequence of bases capable of forming a triple helix or triple strand structure with a portion of particular double stranded DNA, whereby said reporter strand is displaced from said complex upon formation of said triple helix or triple strand structure, and displaced reporter strands are detected to determine the presence of said particular double stranded DNA.
According to the invention there are also provided a DNA probe, a DNA complex, a kit and selected gene sequences, useful for assaying genetic material.
In an embodiment of the invention there also is provided a method of assaying genetic sequences, which comprises contacting a sample containing double stranded DNA to be assayed fixed to a support with an aqueous buffer medium containing a protein which promotes formation of a triple strand structure and containing at least one reporter DNA strand having a detectable label, a portion of the reporter strand consisting of a selected sequence of bases capable of forming a triple helix or triple strand structure with a portion of particular double stranded DNA, incubating the medium, washing to remove unbound reporter, and detecting bound reporter strands to determine the presence of said particular double stranded DNA.
Prior art techniques such as Southern blotting, polymerase chain reaction or ligase chain reaction involve many time-consuming steps. Also since all the steps in the process are performed in the diagnostic laboratory, the staff have to be highly skilled and trained scientists. In the present embodiment, the assay vessel would be supplied containing the preformed reporter/anchor hybrid duplex. In the diagnostic laboratory, the sample DNA is added to the reaction vessel and incubated at a controlled temperature for a defined period of time. These parameters are defined in the production laboratory. The amount of reporter released is then determined using technology not part of this invention. Thus the steps required are simple and rapid and do not require great technical skill on the part of the operator. As a result, the time taken for the assay is significantly reduced and the cost correspondingly reduced.